HPLC Sample and Instrument Preparation

HPLC Sample Preparation

Sample preparation is an important step in LC analysis as it greatly influences the reliability of results. Sample extraction makes the matrix more suitable for analysis, ideally the sample should be prepared with the solvent used for the mobile phase. If a gradient method is being used, the sample should be prepared using the same composition as the start of the method.

Preparation may also be required to either dilute or concentrate the sample, bringing the concentration of analyte within the limits of quantification. The sample should dissolve fully into the solvent used for the mobile phase, split or broadened peaks may be seen if the sample is not fully dissolved.

HPLC Instrument Preparation

Before analysis starts, several points should be checked to assure the proper function of the LC. Firstly, the lamp should be turned on for some time for equilibration purposes. Flushing of the system is a must to get rid of any air within the tubing you may also set the flow to a rate of 1 ml/min to check for leaks. If a fluctuation in the pressure or flow is observed, purge the system using the Purge function within the Maintenance Tool. As a last step, the column must be equilibrated as well, by continuing the flow. As a rule, for a proper equilibration of the column, flush the column with 10 to 20 times the column volume.

Instrument Checklist

HPLC Instrument Start Up 

  • Check for leaks.
  • Turn on detector. Heat lamp.
  • Purge the system with the freshly made mobile phase.
  • Check for air bubbles within the tube and syringe.
  • Equilibrate column.


Instrument Shut Down 

  • Flush column to remove contaminants.
  • If needed, disconnect the column and attach a backpressure device.
  • Flush system with water if buffer was used.
  • Replace mobile phase with 100% Methanol or Isopropanol.
  • Purge pump with a flow rate of 1 ml/min (no column installed).
  • Wash the needle 2-3 times.
  • Turn off the power of each individual module.

Further Assistance

If you require any assistance with your HPLC, please get in touch.